ANA HEp-2 Test System

ANA HEp-2 Test System
REF
IVD
FA2401EB
INTENDED USE
The ZEUS IFA ANA HEp-2 Test System is a pre-standardized assay designed for the qualitative and semi-quantitative detection of antinuclear antibodies. The test is
intended to aid in determining SLE and differentiating clinically similar connective tissue disorders, and is for In Vitro diagnostic use.
SIGNIFICANCE AND BACKGROUND
The indirect fluorescent antibody (IFA) technique method has been used extensively for detecting the presence of ANA in the sera of patients with systemic lupus
erythematosus (SLE), and other clinically similar connective tissue disorders (1 - 5). In addition, ANA may be associated with numerous drug-induced lupus syndromes
(6 - 7) which clinically mimic the spontaneous form of SLE. The IFA technique was adapted to ANA testing by several investigators (8 - 9) following the basic methods
originally described by Coons (10). ANA are primarily composed of IgG antibodies; however, IgA and IgM ANA may also be detected (11). It is now recognized that
many sources of nuclear material may be employed as a substrate for ANA testing. Although most of the original ANA research was performed using rat or mouse
liver or kidney tissue section substrate, the use of human or animal embryonic tissue cell culture substrates has provided a reliable and easy to interpret alternative
substrate for ANA testing. The HEp-2 cell line is a recommended substrate for detecting centromere antibody, which is highly indicative of the CREST variant of
progressive systemic sclerosis (27). There are several different patterns of nuclear and cytoplasmic immunofluorescence. These various patterns and the basis for
them are as follows:
Homogeneous - Homogeneous or diffuse staining patterns of the nucleus is consistent with autoantibodies to native DNA (nDNA) histones and/or
deoxyribonucleoprotein (DNP) (12, 13). The chromosomes of the mitotic cells (dividing cells) are important indicators of a homogeneous pattern because they will
stain as irregularly shaped masses with more intensely stained outer edges.
Speckled Patterns - The speckled pattern is the most commonly observed ANA pattern. A uniform “true speckled” pattern may be seen with centromere antibodies
in cells not in division. A clumpy speckled pattern may be seen with antibodies to n-RNP, Sm, and SSB/La.
1. Fine speckled pattern, chromosome-negative: Numerous small and uniform points of fluorescence uniformly scattered throughout the nucleus. The nucleoli will
generally appear unstained. The mitotic cells may demonstrate a few speckles in their cytoplasm, but the chromosomes will be negative.
2. Coarse speckled pattern, chromosome-negative: Medium-sized points of fluorescence will be scattered throughout the nuclei with distinct nuclear margins.
Larger-sized points of fluorescence may also be observed; however, they are too numerous and variable in size to be identified as a nucleolar pattern. The
chromosomes in the mitotic cells will be negative.
3. Discrete speckled, chromosome (centromere specificity) positive: The chromosomes will be positive in mitotic cells; in fact, the discrete speckles will only be
clustered in the chromosome mass clearly demonstrating the various stages of mitosis. The centromere pattern has been recognized to be associated with the
CREST syndrome, which is a milder variant of progressive systemic sclerosis (PSS). The centromere pattern will demonstrate discrete and uniform points of
fluorescent speckles scattered throughout the nucleus. Mitotic cells will be positive, demonstrating a clustering of the centromeres in the chromosomes in
different arrangements according to the mitotic stage. Harmon, et al (17) demonstrated that serum samples containing highly monospecific anti-SSA/Ro gave
an IF-ANA test pattern of discrete nuclear speckles on a wide variety of human cells and tumor nuclei. Such serum samples with monospecific anti-SSA/Ro
produced very little cytoplasmic staining of substrate cells. A distinct, large, variable speckled pattern of 3 to 10 large speckles in the nucleus has been
described. These patients with large, variable speckles have undifferentiated rheumatic disease syndromes with IgM antihistone H-3 antibody (18).
Nucleolar Pattern - The nucleolar pattern demonstrates a homogeneous or speckled staining of the nucleolus. This pattern is often associated with a dull,
homogeneous fluorescence in the rest of the nucleus. The chromosomes in the mitotic cells will be negative. The nucleolar pattern suggests autoantibodies to 4 - 6S
RNA. The nucleolar fluorescence will appear as homogeneous, clumped, or speckled, depending on the antigen to which the autoantibody reacts. Antinucleolar
antibodies occur primarily in the sera of patients with scleroderma, systemic lupus erythematosus, Sjögren’s syndrome, or Raynaud’s phenomenon (19).
Peripheral (Rim) - The nuclei stain predominantly at their periphery. The chromosomes of the mitotic cells stain as irregularly shaped masses with more intensely
stained outer edges. This pattern is often seen with autoantibodies to nDNA (3, 14 - 16). If the chromosomes of the mitotic cells are negative, then the pattern would
be suggestive of autoantibodies to the nuclear membrane and not to nDNA, and not reported as a peripheral pattern (see nuclear membrane interpretation below).
Additional Patterns
1. Spindle fiber pattern, chromosome-positive: The spindle fiber pattern is unique to cells undergoing mitosis where only the spindle apparatus fluoresces. This
pattern has a “spider web” appearance extending from the centriole to the centromeres. The pattern is suggestive of autoantibodies to the microtubules and
its significance is unclear; however, an association between the spindle fiber pattern and carpal tunnel syndrome has been suggested.
2. Midbody pattern: The midbody pattern is a densely staining region near the cleavage furrow of telophase cells, that is, in the area where the two daughter cells
separate. The clinical significance of the pattern is unknown; however, the pattern has been recognized in selected patients with systemic sclerosis.
3. Centriole pattern: The centriole pattern is characterized by two distinct points of fluorescence in the nucleus of the mitotic cells or one distinct point of
fluorescence in the resting cell. The significance of this pattern is not known; however, it has been observed in PSS.
4. Proliferating cell nuclear antigen (PCNA) pattern: The proliferating cell nuclear antigen pattern is observed as a fine to course nuclear speckling in 30 - 60% of
the cells in interphase, and a negative staining of the chromosome region of mitotic cells. The PCNA is very specific for patients with SLE but not detected in
other connective tissue disease disorders. It has been reported that SLE patients with the PCNA pattern have a higher incidence of diffuse glomerulonephritis.
5. Antinuclear membrane (nuclear laminae): The antinuclear membrane pattern appears as a rim around the nucleus and resembles a rim pattern; however, it is
distinguished from the rim pattern by the fact that the metaphase chromosome stage is negative. This autoantibody is important to report because it is
recognized to be associated with autoimmune liver disease.
Cytoplasmic Patterns
1. Mitochondrial (AMA) pattern: The pattern will characteristically have numerous cytoplasmic speckles with the highest concentration in the peri-nuclear area.
The pattern can be observed in interphase and mitotic cells. The clinical significance of AMA is most frequently an association with primary biliary cirrhosis,
especially when the AMA is a high titer.
2. Golgi apparatus pattern: The golgi apparatus pattern is characterized by positive cytoplasmic staining that is concentrated on only one side of the perinuclear
region. The clinical significance is uncertain, but reports in the literature have suggested an association with SLE and Sjögren’s Syndrome.
3. Lysosomal pattern: The lysosomal pattern is observed as a few discrete speckles sparsely spaced throughout the cytoplasm. The pattern is observed in the
cytoplasm of interphase and mitotic cells. The clinical significance is unknown.
4. Ribosomal pattern: The ribosomal pattern is characterized by numerous cytoplasmic speckles with the highest concentration around the nucleus. It is
distinguished from the mitochondrial pattern because of the smaller specks and higher density. The significance of the pattern is unknown.
5. Cytoskeletal pattern: The cytoskeletal pattern is characterized by a distinct “spider web” or fibrous appearance throughout the cell. It has been reported to be
associated with autoimmune liver disease (anti-smooth muscle).
ZEUS IFA ANA HEp-2 (FA2401EB) Test System
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(Rev. Date 10/15/2014)
ANA Negative
Autoantibody to SSA/Ro is present in high frequency in a clinical subset of lupus called Subacute Cutaneous Lupus Erythematosus (SCLE). Many patients with SCLE
have been falsely labeled as having “ANA-negative” lupus. Many of these so-called “ANA-negative” LE patients will demonstrate a positive IF-ANA on substrate of
HEp-2 cells containing the SSA/Ro antigen (20). Anti-SSA/Ro antibodies may be present in the absence of traditional ANAs, with SLE seen in persons genetically
deficient in C4 and occasionally other complementary deficiencies (21, 22). In addition, C4 deficiency may be associated with increased susceptibility to development
of SLE upon treatment with hydralazine (25). These patients, if female, are likely to deliver infants with congenital heart block or lupus dermatitis (26). Although the
level of ANA may not correlate with the clinical course of a particular autoimmune disease state (6), the various patterns of nuclear staining may be associated with
specific disease states (3, 16, and 28 - 31).
The following table summarizes the various auto-antibodies noted above with respect to disease association:
Table 1
Antibody
Disease State
Relative Frequency of Antibody Detection %
Anti-Jo-1
Myositis
25 - 44% (18)
Anti-Sm
SLE
30 *
Anti-RNP
MCTD, SLE
100** and > 40, respectively
Anti-SSA/Ro
SLE, Sjögren’s
15 and 30 - 40, respectively
Anti-SSB/La
SLE, Sjögren’s
15 and 60 - 70, respectively
Anti-Scl-70
Systemic sclerosis
20 - 28 *
* Highly Specific
** Highly Specific when present alone at high titer
PRINCIPLE OF THE ASSAY
The ZEUS IFA ANA HEp-2 Test System is designed to detect the presence of circulating ANA in human sera. The assay employs tissue cell culture substrate and goat
anti-human immunoglobulin adjusted for optimum use and free of nonspecific background staining. The reaction occurs in two steps:
1. Step one is the sample incubation where any ANA present in the patient sample may bind to the cell substrate, forming an antigen-antibody complex. Other
serum components are subsequently washed away.
2. Step two is the Conjugate incubation where anti-human immunoglobulin labeled with FITC is allowed to react with any human immunoglobulin that bound to
the substrate during the sample incubation. This will form a stable antigen-antibody-conjugate complex at the location where the initial patient antibody bound
to the cell substrate. Excess Conjugate is subsequently washed away. The results of the assay can be visualized using a properly equipped fluorescent
microscope. Any positive reactions will appear as apple-green fluorescent staining within the cell. If the sample had no specific ANA, there will be no distinct
nuclear staining of the cell.
TEST SYSTEM COMPONENTS
Materials Provided:
Each Test System contains the following components in sufficient quantities to perform the number of tests indicated on the packaging label. NOTE: Conjugate and
Controls contain a combination of Proclin (0.05% v/v) and Sodium Azide (<0.1% w/v) as preservatives. SAVe Diluent® contains Sodium Azide (<0.1% w/v) as a
preservative.
● ● ●
1.
ANA HEp-2 Substrate Slides: Ten, 12-well Slides with absorbent blotter and desiccant pouch.
CONJ
2.
Conjugate: Goat anti-human immunoglobulin labeled with fluorescein isothiocyanate (FITC). Contains phosphate buffer with BSA. One, 3.5mL,
amber-capped, bottle.
CONTROL
+
3.
Positive Control (Human Serum): Will produce positive apple-green, homogeneous, staining of the cell nucleus. One, 0.5mL, red-capped, vial.
CONTROL
-
4.
Negative Control (Human Serum): Will produce no detectable nuclear staining. One, 0.5mL, green-capped, vial.
DIL
SPE
5.
SAVe Diluent®: Two, 30mL, green-capped, bottles containing phosphate-buffered-saline. Ready to use. NOTE: The SAVe Diluent® will change
color when combined with serum.
BUF
PBS
6.
Phosphate-buffered-saline (PBS): pH 7.2 ± 0.2. Empty contents of each buffer packet into one liter of distilled or deionized water. Mix until all salts
are thoroughly dissolved. Four packets, sufficient to prepare 4 liters.
MNTMED
7.
Mounting Media (Buffered Glycerol): Two, 3.0mL, white-capped, dripper tipped vials.
EBCS
8.
Evans Blue Counterstain: One, 3.0mL, white-capped, dripper tipped vial.
NOTES:
1.
2.
1.
2.
3.
4.
5.
6.
The following components are not Test System Lot Number dependent and may be used interchangeably with the ZEUS IFA Test Systems, as long as the
product numbers are identical: SAVe Diluent® (Product #: FA005CC), Mounting Media (Product #: FA0009S), and PBS (Product #: 0008S).
Test System also contains:
a. Component Label containing lot specific information inside the Test System box.
b. A CD containing all ZEUS IFA Product Inserts, providing instructions for use.
PRECAUTIONS
For In Vitro diagnostic use.
Follow normal precautions exercised in handling laboratory reagents. In case of contact with eyes, rinse immediately with plenty of water and seek medical
advice. Wear suitable protective clothing, gloves, and eye/face protection. Do not breathe vapor. Dispose of waste observing all local, state, and federal laws.
The wells of the Slide do not contain viable organisms. However, consider the Slide potentially bio-hazardous materials and handle accordingly.
The Controls are potentially bio-hazardous materials. Source materials from which these products were derived were found negative for HIV-1 antigen, HBsAg
and for antibodies against HCV and HIV by approved test methods. However, since no test method can offer complete assurance that infectious agents are
absent, these products should be handled at the Bio-safety Level 2 as recommended for any potentially infectious human serum or blood specimen in the
Centers for Disease Control/National Institutes of Health manual “Biosafety in Microbiological and Biomedical Laboratories”: current edition; and OSHA’s
Standard for Bloodborne Pathogens (20).
Adherence to the specified time and temperature of incubations is essential for accurate results. All reagents must be allowed to reach room temperature (20
- 25°C) before starting the assay. Return unused reagents to their original containers immediately and follow storage requirements.
Improper washing could cause false positive or false negative results. Be sure to minimize the amount of any residual PBS, by blotting, before adding Conjugate.
Do not allow the wells to dry out between incubations.
ZEUS IFA ANA HEp-2 (FA2401EB) Test System
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(Rev. Date 10/15/2014)
21.
22.
The SAVe Diluent®, Conjugate, and Controls contain Sodium Azide at a concentration of <0.1% (w/v). Sodium Azide has been reported to form lead or copper
azides in laboratory plumbing which may cause explosions on hammering. To prevent, rinse sink thoroughly with water after disposing of solution containing
Sodium Azide. This preservative may by toxic if ingested.
Dilution or adulteration of these reagents may generate erroneous results.
Never pipette by mouth. Avoid contact of reagents and patient specimens with skin and mucous membranes.
Avoid microbial contamination of reagents. Incorrect results may occur.
Cross contamination of reagents and/or samples could cause erroneous results.
Reusable glassware must be washed and thoroughly rinsed free of all detergents.
Avoid splashing or generation of aerosols.
Do not expose reagents to strong light during storage or incubation.
Allowing the slide packet to equilibrate to room temperature prior to opening the protective envelope will protect the wells and blotter from condensation.
Collect the wash solution in a disposal basin. Treat the waste solution with disinfectant (i.e.:10% household bleach - 0.5% Sodium Hypochlorite). Avoid
exposure of reagents to bleach fumes.
Do not expose any of the reactive reagents to bleach-containing solutions or to any strong odors from bleach-containing solutions. Trace amounts of bleach
(Sodium Hypochlorite) may destroy the biological activity of many of the reactive reagents within this Test System.
Do not apply pressure to slide envelope. This may damage the substrate.
The components of this Test System are matched for optimum sensitivity and reproducibility. Reagents from other manufacturers should not be interchanged.
Follow Package Insert carefully.
Unopened/opened components are stable until the expiration date printed on the label, provided the recommended storage conditions are strictly followed.
Do not use beyond the expiration date. Do not freeze.
Evans Blue Counterstain is a potential carcinogen. If skin contact occurs, flush with water. Dispose of according to local regulations.
Do not allow slides to dry during the procedure. Depending upon lab conditions, it may be necessary to place slides in a moist chamber during incubations.
1.
2.
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4.
5.
6.
7.
8.
9.
10.
11.
Small serological, Pasteur, capillary, or automatic pipettes.
Disposable pipette tips.
Small test tubes, 13 x 100mm or comparable.
Test tube racks.
Staining dish: A large staining dish with a small magnetic mixing set-up provides an ideal mechanism for washing Slides between incubation steps.
Cover slips, 24 x 60mm, thickness No. 1.
Distilled or deionized water.
Properly equipped fluorescence microscope.
1 Liter Graduated Cylinder.
Laboratory timer to monitor incubation steps.
Disposal basin and disinfectant (i.e.: 10% household bleach – 0.5% Sodium Hypochlorite).
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20.
MATERIALS REQUIRED BUT NOT PROVIDED
The following filter systems, or their equivalent, have been found to be satisfactory for routine use with transmitted or incident light darkfield assemblies:
Transmitted Light
Light Source: Mercury Vapor 200W or 50W
Excitation Filter
Barrier Filter
Red Suppression Filter
KP490
K510 or K530
BG38
BG12
K510 or K530
BG38
FITC
K520
BG38
Light Source: Tungsten – Halogen 100W
KP490
K510 or K530
BG38
Excitation Filter
KP500
FITC
KP500
FITC
1.
2.
3.
Incident Light
Light Source: Mercury Vapor 200, 100, 50 W
Dichroic Mirror
Barrier Filter
TK510
K510 or K530
TK510
K530
Light Source: Tungsten – Halogen 50 and 100 W
TK510
K510 or K530
TK510
K530
Red Suppression Filter
BG38
BG38
BG38
BG38
SPECIMEN COLLECTION
ZEUS Scientific recommends that the user carry out specimen collection in accordance with CLSI document M29: Protection of Laboratory Workers from
Occupationally Acquired Infectious Diseases. No known test method can offer complete assurance that human blood samples will not transmit infection.
Therefore, all blood derivatives should be considered potentially infectious.
Only freshly drawn and properly refrigerated sera obtained by approved aseptic venipuncture procedures with this assay (30, 31). No anticoagulants or
preservatives should be added. Avoid using hemolyzed, lipemic, or bacterially contaminated sera.
Store sample at room temperature for no longer than 8 hours. If testing is not performed within 8 hours, sera may be stored between 2 - 8°C, for no longer
than 48 hours. If delay in testing is anticipated, store test sera at –20°C or lower. Avoid multiple freeze/thaw cycles which may cause loss of antibody activity
and give erroneous results. It is the responsibility of the individual laboratory to use all available references and/or its own studies to determine stability criteria
for its laboratory (38).
STORAGE CONDITIONS
Unopened Test System.
Mounting Media, Conjugate, SAVe Diluent®, Slides, Positive and Negative Controls, and Evans Blue Counterstain.
Rehydrated PBS (Stable for 30 days).
Phosphate-buffered-saline (PBS) Packets.
ZEUS IFA ANA HEp-2 (FA2401EB) Test System
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(Rev. Date 10/15/2014)
ASSAY PROCEDURE
Remove Slides from refrigerated storage and allow them to warm to room temperature (20 - 25°C). Tear open the protective envelope and remove Slides. Do
not apply pressure to flat sides of protective envelope.
2. Identify each well with the appropriate patient sera and Controls. NOTE: The Controls are intended to be used undiluted. Prepare a 1:40 dilution (e.g.: 10µL of
serum + 390µL of SAVe Diluent® or PBS) of each patient serum. The SAVe Diluent® will undergo a color change confirming that the specimen has been
combined with the Diluent.
Dilution Options:
a.
As an option, users may prepare initial sample dilutions using PBS, or Zorba-NS (Zorba-NS is available separately. Order Product Number FA025 – 2, 30mL
bottles).
b. Users may titrate the Positive Control to endpoint to serve as a semi-quantitative (1+ Minimally Reactive) Control. In such cases, the Control should be
diluted two-fold in SAVe Diluent® or PBS. When evaluated by ZEUS Scientific, an endpoint dilution is established and printed on the Positive Control vial (±
one dilution). It should be noted that due to variations within the laboratory (equipment, etc.), each laboratory should establish its own expected endpoint titer for each lot of Positive Control.
c.
When titrating patient specimens, initial dilutions should be prepared in SAVe Diluent®, PBS or Zorba-NS and all subsequent dilutions should be prepared
in SAVe Diluent® or PBS only. Titrations should not be prepared in Zorba-NS.
3. With suitable dispenser (listed above), dispense 20 - 40µL of each Control and each diluted patient sera in the appropriate wells.
4. Incubate Slides at room temperature (20 - 25°C) for 20 - 30 minutes.
5. Gently rinse Slides with PBS. Do not direct a stream of PBS into the test wells.
6. Wash Slides two, additional time, changing PBS between washes. Slides may soak during each wash for up to five minutes. NOTE: For those using automated
washers, set the washer to wash each well three times with a soak of zero to five minutes.
7. Remove Slides from PBS one at a time. Invert Slide and key wells to holes in blotters provided. Blot Slide by wiping the reverse side with an absorbent wipe.
CAUTION: Position the blotter and Slide on a hard, flat surface. Blotting on paper towels may destroy the Slide matrix. Do not allow the Slides to dry during
the test procedure.
8. Add 20 - 40µL of Conjugate to each well.
9. Repeat steps 4 through 7. If desired, user may add Evans Blue Counterstain to the wash PBS during the second 5 minute wash interval. Use 3 - 5 drops of Evans
Blue per 150mL of PBS.
10. Apply 3 - 5 drops of Mounting Media to each Slide (between the wells) and coverslip. Mounting Media must be added within two hours of completing the last
wash cycle. Examine Slides immediately with an appropriate fluorescence microscope.
NOTE: If delay in examining Slides is anticipated, seal coverslip with clear nail polish and store in refrigerator. It is recommended that Slides be examined on the
same day as testing.
1.
QUALITY CONTROL
1.
2.
Every time the assay is run, a Positive Control, a Negative Control, and a Buffer Control must be included.
It is recommended that the Controls be read prior to evaluating the test samples. If the Controls do not appear as described, results may be invalid.
a.
Negative Control - characterized by the absence of specific fluorescence and a red, or dull green, background staining of all cells due to counterstain.
b. Positive Control (homogeneous pattern) - characterized by apple-green fluorescence. The homogeneous staining pattern is a diffused uniform staining of
the entire nucleus.
3. Additional Controls may be tested according to guidelines or requirements of local, state, and/or federal regulations or accrediting organizations.
NOTE:
a. Non-specific reagent trapping may exist. It is important to adequately wash Slides to eliminate false positive results.
b. The intensity of the observed fluorescence may vary with the microscope and filter system used.
c.
Non-nuclear staining of the cell substrate may be observed with some human sera.
1.
2.
3.
4.
INTERPRETATION OF RESULTS
The interpretation of the results depends on the pattern observed, the titer of the autoantibody, and the age of the patient. The elderly, especially women, are
prone to develop low-titered autoantibodies (<1:80) in the absence of clinical autoimmune disease. In contrast, a 1:20 titer of a significant pattern of
autoantibody(s) in a young person may suggest that overt disease may occur later. Experience suggests that a 1:40 dilution is a good dilution to screen for ANA.
Low-titer positive results may occur in apparently healthy persons; therefore, the ANA results must always be interpreted in light of the patient’s total clinical
presentation.
Titers less than 1:40 are considered negative.
Positive test: A positive reaction is the presence of any pattern of nuclear apple-green staining observed at a 1:40 dilution based on a 1+ to 4+ scale of staining
intensity. 1+ is considered a weak reaction and 4+ a strong reaction. All sera positive at 1:40 should be titered to endpoint dilution. This is accomplished by
making 1:40, 1:80, 1:160, etc. serial dilutions of all positives. The endpoint titer is the highest dilution that produces a 1+ positive reaction.
Homogeneous patterns with peripheral accentuation are frequently found in sera from patients with SLE.
Pattern
Homogeneous:
High Titer
Low Titer
Centromere
Speckled
Nucleolar
Peripheral
1.
2.
3.
4.
5.
Disease Most Frequently Found In
Reference
SLE
Rheumatoid Arthritis and other diseases
CREST Syndrome variant of PSS
Scleroderma, Raynaud’s Syndrome, Sjögren’s Syndrome,
Mixed connective tissue disease
Scleroderma
SLE
(3, 8, 9, and 16)
(1)
(27)
(34 - 36)
(37)
(2, 8, 9, and 16)
LIMITATIONS OF THE ASSAY
The ZEUS IFA ANA HEp-2 Test System is a laboratory diagnostic aid and by itself is not diagnostic. Positive ANA may be found in apparently healthy individuals.
It is therefore imperative that ANA results be interpreted in light of the patients clinical condition by a medical authority.
SLE patients undergoing steroid therapy may have negative test results.
Many commonly prescribed drugs may induce ANA (6, 7).
One autoantibody pattern may partially or completely obscure the diagnostic features of the other. In such instances, it is necessary to titrate the serum.
No definitive association between the pattern of nuclear fluorescence and any specific disease state is intended with this product.
EXPECTED RESULTS
The expected value in the normal population is negative or less than 1:40. However, apparently healthy individuals may contain ANA in their sera (36). This
percentage increases with aging, particularly in the 7th decade of life.
ZEUS IFA ANA HEp-2 (FA2401EB) Test System
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(Rev. Date 10/15/2014)
PERFORMANCE CHARACTERISTICS
The ZEUS IFA ANA HEp-2 Test System was tested in parallel with a reference procedure employing rat liver substrate. Routine ANA testing was performed by both
procedures on 434 patient specimens. Of these 434 sera, 116 were positive by both procedures. The ZEUS IFA ANA HEp-2 Test System showed 97% agreement with
respect to positive and negative results, and 100% with respect to staining pattern. Of the 21 discrepancies in titer, the ZEUS IFA ANA HEp-2 procedure was one
dilution lower in 18 specimens. Five of these 18 specimens that were negative using the ZEUS IFA ANA HEp-2 procedure were positive at 1:20 by the rat liver
reference procedure.
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